1. Paired-pulse synaptic stimulation of hippocampal neurons in microcultures resulted in depression of synaptic currents mediated by both NMDA and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors. However, NMDA EPSCs were more severely depressed than AMPA EPSCs. 2. Partial NMDA receptor blockade reduced paired-pulse depression of NMDA but not of AMPA synaptic currents while partial AMPA receptor blockade had no effect on paired-pulse depression of AMPA EPSCs. These results suggest that ion flux through NMDA receptors is important in paired-pulse depression of NMDA responses but has no effect on AMPA responses. 3. Low extracellular Ca2+ concentrations or positive postsynaptic holding potentials reduced paired-pulse depression of NMDA EPSCs to near that of AMPA responses. 4. Brief paired applications of exogenous glutamate to neurons produced Ca(2+)-dependent depression similar to the depression of NMDA synaptic responses and synaptic stimulation depressed responses to exogenously applied NMDA. 5. Physiological concentrations of Mg2+ prevented expression of the postsynaptic modulation of NMDA EPSCs at -70 mV, but partial relief of Mg2+ block of the NMDA channel with depolarization increased paired-pulse depression of NMDA EPSCs.